PosterREPRINT URINE SAMPLES FOR GLUCURONIDES BY LC/MS/MS Andrew G. Baker*, Nicholas J. Ellor, Jennifer L. Jones; Waters Corporation, MS Technology Center, 100 Cummings Center, Suite 407N, Beverly, MA 01915 ABSTRACT The traditional study of in vivo drug metabolism in o o plasma or urine samples is often complicated by the presence of many endogenous compounds. Several mass spectrometric techniques are often applied in drug metabolism studies. Conjugation reactions, particularly gl ucuronidation, can be studied using either the neutral loss or precursor ion experiment, however the experiments are not usually done at the same time. Using a hybrid quadrupole/time of flight instrument, a new method of performing the neutral loss and precursor ion scan experiments simultaneously has been devised. Using this novel approach, which is more sensitive and selective than the corresponding triple quadrupole experiments, several Phase 1 and Phase 2 metabolites of diphenhydramine were identified from a human urine sample collected 4 hours after drug administration. Accurate mass measurements were made in the same acquisition, and elemental compositions of the found metabolites were assigned to within 5 ppM. Diphenhydramine was extensively metabolized to form a numb er of excreted metabolites, including N-demethylation, aryl, and N-hydroxy metabolites. Glucuronide conjugation was observed for several of the hydrox-metabolites as well as the N-glucuronide. INTRODUCTION Mass spectrometry is a well-established technique used in many ways throughout the drug discovery and development process. Profiling both in vitro samples o o for initial metabolic information and in vivo samples o o such as plasma, bile, or urine for a more complete description of drug metabolism is one such area. As the pharmaceutical life cycle evolves, metabolism studies are often brought into the discovery phase to reject compounds with poor metabolic profiles or toxicity issues earlier in the timeline. Synthesis of radioactive analogues for metabolism studies is not feasible, thus other, more selective techniques must be used. Conjugation reactions such as glucuronidation or sulfation serve to increase the polarity of a drug or metabolite, making excretion more likely. These Phase 2 metabolites are often studied using the classic triple quadrupole neutral loss experiment. This experiment, while well suited for selectively detecting conjugated metabolites from complex samples such as urine or bile cannot be used to assay several metabolic processes at once, as the quadrupoles are scanned with a mass offset corresponding to the conjugate. To determine another conjugation, either a loss in duty cycle from alternating scans with different mass offsets or another LC/MS acquisition is required. Two novel experiments using a hybrid quadrupole- time of flight Q-Tof ™ instrument have been devised to overcome this limitation in duty cycle. 1 As this experiment is performed using the Q-Tof, the resulting accurate mass MS/MS spectrum is particularly useful for structural elucidation. Diphenhydramine is a well characterized Histamine H1 receptor antagonist. The major urinary metabolites include a N + glucuronide, N-Oxide, as well as several minor metabolites. 2,3,4 This model system was used to demonstrate the application of these two novel Q-Tof experiments.
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BY LC/MS/MS
Andrew G. Baker*, Nicholas J. Ellor, Jennifer L. Jones; Waters Corporation, MS Technology Center, 100 Cummings Center, Suite 407N, Beverly, MA 01915
ABSTRACT
The traditional study of in vivo drug metabolism in in vivo drug metabolism in in vivoplasma or urine samples is often complicated by the presence of many endogenous compounds. Several mass spectrometric techniques are often applied in drug metabolism studies. Conjugation reactions, particularly glucuronidation, can be studied using either the neutral loss or precursor ion experiment, however the experiments are not usually done at the same time. Using a hybrid quadrupole/time of flight instrument, a new method of performing the neutral loss and precursor ion scan experiments simultaneously has been devised.Using this novel approach, which is more sensitive and selective than the corresponding triple quadrupole experiments, several Phase 1 and Phase 2 metabolites of diphenhydramine were identified from a human urine sample collected 4 hours after drug administration.Accurate mass measurements were made in the same acquisition, and elemental compositions of the found metabolites were assigned to within 5 ppM. Diphenhydramine was extensively metabolized to form a number of excreted metabolites, including N-demethylation, aryl, and N-hydroxy metabolites. Glucuronide conjugation was observed for several of the hydrox-metabolites as well as the N-glucuronide.
INTRODUCTION
Mass spectrometry is a well-established technique used in many ways throughout the drug discovery and development process. Profiling both in vitro samples in vitro samples in vitrofor initial metabolic information and in vivo samples in vivo samples in vivosuch as plasma, bile, or urine for a more complete description of drug metabolism is one such area. As the pharmaceutical life cycle evolves, metabolism studies are often brought into the discovery phase to reject compounds with poor metabolic profiles or toxicity issues earlier in the timeline. Synthesis of radioactive analogues for metabolism studies is not feasible, thus other, more selective techniques must be used. Conjugation reactions such as glucuronidation or sulfation serve to increase the polarity of a drug or metabolite, making excretion more likely. These Phase 2 metabolites are often studied using the classic triple quadrupole neutral loss experiment. This experiment, while well suited for selectively detecting conjugated metabolites from complex samples such as urine or bile cannot be used to assay several metabolic processes at once, as the quadrupoles are scanned with a mass offset corresponding to the conjugate. To determine another conjugation, either a loss in duty cycle from alternating scans with different mass offsets or another LC/MS acquisition is required. Two novel experiments using a hybrid quadrupole- time of flight Q-Tof™ instrument have been devised to overcome this limitation in duty cycle.1 As this experiment is performed using the Q-Tof, the resulting accurate mass MS/MS spectrum is particularly useful for structural elucidation.
Diphenhydramine is a well characterized Histamine H1 receptor antagonist. The major urinary metabolites include a N+ glucuronide, N-Oxide, as well as several minor metabolites.2,3,4 This model system was used to demonstrate the application of these two novel Q-Tof experiments.
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EXPERIMENTAL
Urine was collected immediately before and 4 hours after administration of a single 25 mg dose of generic diphenhydramine and frozen until analysis. Urine samples were diluted 1:5 with Milli-Q® water prior to analysis. No other sample pretreatment was done.
LC Conditions:
A Waters® 2795 XC Separations Module equipped with a Waters Symmetry® C18 column (2.1 x 150 mm) and 2996 photodiode array detector was used for the chromatographic separation.
Mobile Phases: A= 0.05% Aqueous Trifluoroacetic Acid
Mass Spectrometer: A Waters Q-Tof Ultima™ API-US mass spectrometer equipped with LockSpray™ was operated in positive ion electrospray mode. Prior to use, the instrument was tuned to greater than 10,000 resolution (FWHM) and calibrated using polyalanine. Leucine enkephalin was infused through the reference channel for on-line acquisition of accurate mass measurements.
The offset voltage on the collision cell is modulated between low (5 eV) and high (15 eV) voltages on alternate acquisitions. Ions are selected for subsequent MS/MS acquisition based on the presence of a defined neutral loss between pairs of ions in the low and high collision energy survey acquisitions. Because of the high resolution and high mass accuracy of the TOF mass analyzer, very stringent criteria (10 ppM mass accuracy) can be used to select ions for the MS/MS acquisition.
MetaboLynx™ Processing of Expected and Unexpected Metabolites from Diphenhydramine
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Precursor Ion Discovery Mode
The Q-Tof is operated in the normal fashion, however the offset voltage on the collision cell is modulated between low (5 eV) and high (15 eV) voltages on alternate acquisitions. Only after a fragment ion is found in the high energy survey scan will ions be selected for MS/MS from the low energy survey scan. Because of the high resolution and high mass accuracy of the TOF mass analyzer, very stringent criteria (10 ppM mass accuracy) are used to identify fragment ions that trigger the MS/MS acquisition.
Fast Separation of Diphenhydramine Metabolites from urine using Precursor Ion Discovery Acquisition
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Conclusions• Q-Tof accurate mass MS and MS/MS are useful
for metabolite structural elucidation • Major metabolites of Diphenhydramine--
Diphenhydramine N-Glucuronide and Diphenhydramine N-Oxide identified and characterized
• Novel Precursor Ion Discovery DDA mode with concominant search for neutral losses increases the duty cycle over the classic triple-quadrupole experiments.
• Stringent mass accuracy criteria (10 ppm) for identifying fragment ions or neutral losses enables higher quality results
• Specificity of ENL maintained under fast gradient conditions
References
1. Langridge, J.I., Hoyes, J.B., Bateman, R.H., Millar, A.L., Carruthers, R., Jones, C., and Jensen, O.N., Poster at 49th ASMS 2001.
2. Sharma, A. and Hamelin, B.A. Curr. Drug Metab. 2003, 4, 105-129.
